Ribothrypsis: mechanisms and implications for gene expression regulation PROJECT SUMMARY / ABSTRACT Messenger RNAs transmit the genetic information that dictates protein production and are a nexus for numerous pathways that regulate gene expression. The prevailing view of canonical mRNA decay is that it is mediated by deadenylation and decapping followed by exonucleolysis from the 3' and 5' ends. We recently described ribothrypsis, an endonucleolytic pathway of cotranslational mRNA decay, mediated by ribosome-phased cleavages of the mRNA as it exits the ribosome channel. We posit that ribothrypsis is a unifying and evolutionary conserved mechanism that underlies cotranslational decay of all mRNAs: canonical and aberrant mRNAs that degrade via surveillance mechanisms, such as No-Go Decay (NGD) or Non-Stop Decay (NSD). In that sense, ribothrypsis may be viewed as ?NGD/NSD on steroids?; or conversely NGD/NSD may be viewed as a subset of ribothrypsis. The central integrator of mRNA decay in ribothrypsis is the translating ribosome that under certain conditions activates or recruits an unknown endonuclease (ribothrypsin) to cleave the mRNA as it exits the ribosome. In this proposal, we will investigate the impact of ribothrypsis in gene expression analysis; and mechanisms and impact of ribothrypsis in gene expression regulation. We discovered that endogenously generated mRNA fragments represent a sizable fraction of the total mRNA pool. This finding complicates interpretation of results obtained with all current methods that assay mRNAs, which do not take ribothrypsis into account, and necessitates the development of new experimental and computational tools for RNA sequencing, which we will develop in Aim1. In Aim2, we will investigate ribothrypsis triggers and unexpected roles of ribothrypsis in gene expression regulation via upstream Open Reading Frames (uORFs), and in the decay of long noncoding RNAs (lncRNAs). We will also study molecular mechanisms of ribothrypsis in vitro and in vivo and identify ribothrypsin.